The present invention relates to methods for generating dopaminergic neurons in vitro from embryonic and adult central nervous system cells. Specifically, these cells are isolated, cultured in vitro and stimulated to differentiate into dopaminergic neurons by down-regulating COUP-TFI and/or COUP-TFII expression or increasing NOT1 expression. These newly generated dopaminergic neurons may serve as an excellent source for cell replacement therapy in neurological disorders in which the dopaminergic system is compromised.
The mammalian central nervous system is a highly diversified neural network of cells that form intricate intercellular connections. Once these neural cells are damaged, they do not typically regenerate. Accordingly, treatment of neurological disorders, such as neurodegenerative diseases and neurotrauma, has focused on replacing damaged neural cells with healthy cells. A major obstacle in the field of neuronal transplantation is the inadequacy of donor material. In recent years, therapeutic transplantations have been performed using human fetal tissue as the donor substrate. In addition to the controversial ethical dilemmas that surround the use of human fetal tissue, there are also critical methodological difficulties. Because fetal tissue is obtained from freshly aborted fetuses, there is a limited supply of available tissue. Further, the recipient may immunologically reject the fetal tissue, once transplanted. Moreover, transplanting fresh fetal tissue may result in the transmission of infectious diseases. Even with extensive screening of fetal tissue, certain infections, such as HIV, may not yet be present at clinically significant levels and may go undetected during the screening process. Because of the significant obstacles inherent to fetal tissue transplantation, alternative sources of neuronal cells, especially cells that can be used for autologous transplantation, would be a breakthrough in the field of neuroscience. A renewable source of normal human neural cells would be an indispensable tool in clinical studies of neurotrauma and neurodegenerative diseases. Further, the use of such cells may eliminate the need for fetal human tissue in therapeutic approaches aimed at restoring neurological function by transplantation of nervous system cells.
Central nervous system stem cells represent a renewable source of human neural cells, which may eliminate the need for fetal human tissue in restoring neurological function by intracerbral and intraspinal transplantation. Nervous system stem cells have been isolated from human embryonic and adult brain (Svendsen et al., 1996; Chalmers-Redman et al., 1997). Multipotent stem cells are present in the entire ventricular neuraxis of the adult mammalian central nervous system, including the spinal cord (Morshead and van der Kooy, 1992; Reyolds and Weiss, 1992; Lois and Alvarez-buylla, 1993, 1994; Morshead et al., 1994; Weiss et al., 1996a, b). In response to certain growth factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF), these stem cells can be made to proliferate and differentiate into neuronal cells (neurons) and glial cells (astrocytes and oligodendrocytes) (Reynolds and Weiss, 1992; Morshead et al., 1994; Weiss et al., 1996b).
Although propagation and subsequent transplantation of stem cells derived from adult brain tissue represents an advancement in the field of neurology, methods to differentiate these progenitor cells into specific neuronal populations are required in order to use these cells as a stable source of transplant material. The present invention describes a novel method to induce stem cells to express catecholaminergic neuronal features and, specifically, to produce dopamine. These newly generated dopaminergic neurons may serve as an excellent source for cell replacement therapy in neurological disorders, such as Parkinson""s disease, in which the dopaminergic system is compromised.
In the preferred embodiment of this invention, dopaminergic neurons are generated in vitro by initially isolating human neuronal stem cells from fetal or adult central nervous system tissue. These stem cells are then cultured in the presence of growth factors such as bFGF and leukemia inhibitory factor LIF. The growth factors are subsequently removed and the cells are treated with chemicals that stimulate initial differentiation, such as all-trans retinoic acid and dibutyryl cAMP. The expression of chicken ovalbumin upstream promoter transcription factor (COUP-TF) is then inhibited to induce specific differentiation of dopaminergic neurons. Specifically down-regulating COUP-TFI and/or COUP-TFII expression by using antisense oligonucleotides directed to COUP-TF sequences stimulates differentiation into dopaminergic neurons. It is believed that this differentiation into dopaminergic neurons is due to the increased activity of NOT1 when it is released from inhibition by COUP-TF factors. Thus in another embodiment of the invention neuronal stem cells are stimulated to differentiate into dopaminergic neurons by stimulating NOT1 activity in vitro.